Antioxidant triacylglycerols and lipid compositions

ABSTRACT

A lipid composition includes triacylglycerols that include unsaturated fatty acids such as alpha linolenic acid and antioxidant moeities such as pyruvic acid.

BACKGROUND OF INVENTION

The present invention relates to a novel triacylglycerol incorporatingan antioxidant moiety and lipid compositions comprising triacylglycerolsincluding antioxidant moieties and unsaturated fatty acids.

Fatty acids are saturated or unsaturated aliphatic monocarboxylic acids,usually with an even number of carbon atoms that occur naturally in theform of glycerides in fats and fatty oils, waxes and essential oils.Saturated fatty acids have the general formula, C_(n)H_((2n+1))COOH.Hydroxy-fatty acids such as ricinoleic acid are also known to be presentnaturally in certain oils. All fatty acids are carboxylic acids, whichis a broad term that encompasses any compound with a carboxylic group,COOH. Fatty acids are numbered from the carboxylic carbon atom. Theposition of double bonds is indicated by the Greek letter delta (δ)followed by the carbon number of the double bond, i.e., C 20:5 omega-3 δ5,8,11,14,17. The omega (ω) notation refers to the position of a doublebond as an indicated number of carbon atoms from the non-carboxyterminal end of an unsaturated fatty acid. The designation foreicosapentaenoic acid, an omega-3 polyunsaturated fatty acid, is C 20:5ω-3 δ 5,8,11,14,17. Docosapentaenoic acid is C 22:5 ω-3 δ 7,10,13,16,19and docosahexaenoic acid (DHA) is C 22:6 ω-3 δ 7,10,13,16,19. Thedesignation omega-6 (ω-6) refers to a fatty acid such as linoleic acidthat has a double bond which is at carbon position six from thenon-carboxy terminal end of the fatty acid.

The ω-3 fatty acids are essential for maintenance of health andprevention of disease. As noted above, DHA is a ω-3 fatty acid having 22carbon atoms and three double bonds. It is the major component ofinsulation tissue for the retinal photoreceptors. It plays a major rolein the [1]: • Maintenance of cell membrane fluidity in brain and eyes •Reduction of intraocular pressure • Constant renewal of retinalcomponents after oxidative damage • Reduction of clogging/hardening ofarteries • Enhancement of visual acuity Thus, a supply of DHA, would beuseful in preventing vision disorders such as age-related maculardegeneration and glaucoma through the reduction of arterial plaque andintraocular pressure. In fact, the ω-3 fatty acids cause a lowering ofblood pressure throughout the body's arterial network, reduction incholesterol levels, as well as vasodilation resulting in the alleviationof coronary heart disease.

The ω-3 fatty acids also act as anti-inflammatory agents, making thembeneficial for patients with rheumatoid arthritis and other inflammatoryailments. These acids also protect myelin, which shields the nerves, andmay be useful in treating or ameliorating a variety of neural disorderssuch as dementia or depression. These fatty acids may also be helpful inpreventing cancer. However, due to their high unsaturation, they arequite unstable and subject to oxidation at the ω-3 double bond.

DHA can be supplied to the body either as dietary DHA or in precursorform as alpha-linolenic acid (ALA), also from a dietary source. DietaryDHA originates mainly from fish oils with the associated drawbacks suchas the fishy odor and possible mercury contamination. However, findingsfrom animal studies suggest that brain cells may prefer to make DHA fromits precursor, ALA, rather than absorb it pre-formed [2].

ALA is obtained from plant seed oils such as oils from flaxseed,perilla, hemp, canola or soybean and is relatively odor-free. Acomparison between the properties of ALA and DHA indicates that ALAsupplementation is also superior to that of DHA as a result of manydifferent factors such as:

-   -   1. Price: Cost of dietary DHA supplementation even without        extraction and processing from fish oil is very high.    -   2. Absorption: The pancreatic lipase activity towards fatty        acids decreases with chain length. Consequently, DHA is not as        well accepted as substrate and is released more slowly from        triacylglycerols as compared to ALA.    -   3. Both achieve similar effects: ALA can be converted to DHA to        the extent required by the body even in preterm infants. The        brain/retina also has a capability for the conversion.    -   4. Stability: ALA with its three double bonds is less prone to        oxidative damage than DHA with its six double bonds.    -   5. Concentration: The nervous system is not protected against a        large excess of DHA, which could increase n−3/n−6 ratio and        could lead to abnormal function, and which might be difficult to        reverse. This DHA excess is avoidable by supplying the        precursor, ALA, which will be converted to DHA as required by        the body.

Consequently, it may be preferable to supply DHA through its precursor,ALA, in nutraceutical compositions and products.

It is known that mitochondrial and peroxisomal fatty oxidation ratesincrease with increasing dietary levels of ALA. The activity of enzymessuch as carnitine palmitoyltransferase, acyl-CoA oxidase, 3-ketoacyl-CoAthiolase, and 2,4-dienoyl-CoA reductase is enhanced. Smaller butsignificant increases by ALA of the activity of acyl-CoA dehydrogenase,enoyl-CoA hydratase, and delta 3, delta 2-enoyl-CoA isomerase have alsobeen observed. However, dietary ALA was reported to greatly reduce theactivities of some enzymes such as 3-hydroxy-acyl-CoA dehydrogenase andpyruvate kinase. Therefore, while β-oxidation of fatty acids is higherfor unsaturated fats, the activity of pyruvate kinase (which convertsglucose catabolite, phosphoenol pyruvate into pyruvate) an importantenzyme in the glycolysis pathway is reduced, implying lesser energy. Anexogenous supply of pyruvic acid will also enable further stimulation ofthe fatty acid anabolic pathways to convert them into their moreunsaturated counterparts rather than their channeling towardsβ-oxidation.

Hence, it may be beneficial to supplement ALA with pyruvic acid tooffset this effect. Pyruvic acid is a three-carbon alpha-keto acid withrelatively strong antioxidative activity and may enhance the stabilityof ALA when combined in a formulation. However, pyruvic acid is astrong, unstable ketoacid, which cannot be administered orally orparenterally. Salts of pyruvic acid are also not physiologicallysuitable. Amino-compounds containing pyruvate such as pyruvylglycinelead to excessive nitrogen loads. Also, flooding plasma with glycine mayinterfere with the transport of some amino acids across the blood-brainbarrier. Accordingly, these pyruvate compounds are less suited totreating an organ in vivo, and it is recognized that a need exists toprovide a pyruvate delivery compound that is more physiologicallyacceptable [3].

One such potential compound is a pyruvylglycerol. This would be morestable and pH-neutral. The acylglycerol will be hydrolyzed to pyruvicacid by non-specific esterases present in plasma, tissues and thegastrointestinal tract as well as gastric and pancreatic lipases, andsubsequently neutralized by the body's buffers.

Apart from the benefits of alpha-keto acids described above, they alsoconfer other health benefits particularly due to their antioxidantcapacity. This is possible because they can act as scavengers of freeradicals and can also prevent lipid peroxidation by inhibiting formationof free radicals. This ensures their usefulness in prevention andtreatment of disorders related to aging (such as cataract and glaucoma),diabetes, calcium overexcretion (as in osteoporosis) and cancers.

Structured lipids consisting of fatty acids esterified to the glycerylbackbone are known in the art. Structured triacylglycerols, in contrastto natural triacylglycerols, can be classified as any oil and fatmodified or synthesized by any artificial means, such as hydrogenation,fractionation, blending, interesterification, esterification, and evenfrom bioengineered plants. However, the common definition of structuredtriacylglycerols refers to those oils and fats containingpolyunsaturated fatty acids and medium- or short-chain fatty acids, orthose in which different fatty acids are specifically located in theglycerol backbone.

Most structured lipids have involved the interesterification of oils torearrange the fatty acid distribution, to enhance either the content ofPUFA or that of the medium-chain (MCFA—number of carbons=8, 10, 12) orshort-chain fatty acids (SCFA—number of carbons=3, 4, 6). MCFA and SCFAare excellent sources of ready energy for the body due to their rapidabsorption and are preferred because they have a low caloric value.Structured lipids containing MCFA are well known and their synthesis andcomposition have been widely reported [4-16].

Commercially available structured triacylglycerols include Salatrim™(Benefat™ containing acetic, propionic, butyric and stearic acidsesterified to glycerol) from Cultor Food Science and Nabisco Inc.,Caprenin™ (caprocaprylobehenin, containing caprylic, capric and behenicacids esterified to glycerol) from Procter & Gamble, Captex™ from AbitecCorp., Neobee™ from Stepan Company, Impact™ from Novartis Nutrition, andStructolipid™ from Fresenius Kabi. Enzymatic interesterification withsn-1,3 specific lipases has also been used by industry for theproduction of structured lipids usable as cocoa butter substitutes[17-20].

Although the prior art shows that there has been relatively high degreeof activity in designing triglycerides [21-36] for certain nutritionaland medical uses, there is no teaching in any of the prior art, whichrefers to combining antioxidant short chain alpha-keto carboxylic acidswith long-chain unsaturated fatty acids.

Pyruvate compounds such as pyruvate thiolester, glycerol-pyruvate esteror a dihydoxyacetone-pyruvate ester have been synthesized [37-39]. Theglycerol-pyruvate esters synthesized were pyruvyl-diacetyl-glycerol anddipyruvyl-acetyl-glycerol. These were prepared by esterification ofdiacetin and monoacetin, respectively, with pyruvyl chloride. It appearsthat these are the only glyceryl pyruvate compounds reported inliterature, whereby pyruvic acid is coesterified with a short-chainfatty acid such as acetic acid.

However, there is no suggestion or disclosure of a triacylglycerol or anoil containing pyruvic or other α-keto acids with widespread healthbenefits, and hence, for use as a nutraceutical or functional foodbioactive.

Therefore, there is a need in the art for lipid composition, whichincludes triacylglycerols comprising an antioxidant moiety andnutritionally beneficial fatty acids and methods for forming such alipid composition with relatively high yields.

SUMMARY OF INVENTION

The present invention provides a novel lipid composition, which deliversessential or beneficial fatty acids in an optimal manner with respect tostability and biosorptivity. Stability of the composition is enhanced bythe inclusion of an antioxidant esterified to the glycerol backbone,preferably in a bioavailable manner. This lipid composition is useful asa nutraceutical as well as functional food ingredient. In this respect,the properties of the lipid are useful as it does not give rise toundesirable odors or flavors in food formulations, and does not havemuch impact on the physical properties of the functional food.

Lipid compositions of the present invention comprise triacylglycerolscomprising fatty acids known to confer health benefits on their own orthrough their metabolic products, with respect to preventing oralleviating disorders/symptoms related to aging, inflammatoryconditions, coronary heart disease, cancer and calcium retention. Thefatty acid constituents are preferably essential or beneficial fattyacids, which are intrinsically stabilized by an antioxidant moeity thatis part of the molecule. These compositions can be formulated asnutraceuticals and functional food ingredients.

The novel triacylglycerols of the present invention comprise ω-3 fattyacids as well as an antioxidant esterified to the glycerol backbone.Also present are other 16- and 18-carbon saturated and unsaturated fattyacids, so as to yield a composition with the essential fatty acids in anoptimally desired n−3: n−6 fatty acid ratio of greater than about 2,preferably greater than about 2.5 and more preferably greater than about3. This composition possesses a high degree of unsaturation, stabilizedby the antioxidant, which also functions as a metabolic complement tothe n−3 fatty acids. This structured lipid may assist in the preventionor treatment of diseases resulting from inflammation, oxidative stress(as in aging), cancers; and may assist in maintaining the health of thecirculatory and nervous systems owing to the presence of essential (n−3)fatty acids which give rise to the biologically important fatty acidssuch as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Theantioxidant contributes by reducing oxidative stress, as well as byoffsetting inhibition of energy-yielding pathways which occurs as aresult of n−3 fatty acid metabolism. It is also a useful source ofinstant energy and therefore enhances the nutritional effects of thelipid composition. The lipid can be used as a dietary supplement or asthe functional ingredient of functional foods such as cultured dairyproducts, confectioneries and nutritional bars and beverages.

It is believed that unsaturated fatty acids are also best absorbed inform of acylglycerols [40]. Hence, one preferred embodiment of thisinvention is a triacylglycerol with ALA and pyruvic acid as the majoracyl components. Since the gastric and pancreatic lipases have aregioselectivity for the sn-1 and sn-3 hydroxyl positions on theglycerol backbone [41], the target compound should preferably havepyruvic acid esterified at one or both these positions. As the n−3:n−6ratio is just as important as the absolute levels of ALA, the targetcompound may also contain small amounts of other fatty acids such aslinoleic and oleic acids [42].

Therefore, in one aspect, the invention comprises a triacylglycerolcomprising an antioxidant moiety, which is preferably an alpha-ketoacid, and more preferably, pyruvic acid. The pyruvic acid may beesterified to any of the 3 carbons of the glycerol backbone, however, itis preferred that the pyruvic acid be esterified at the sn-1 or sn-3positions. The remaining two fatty acids may be saturated or unsaturatedand of any chain length. Preferably, at least one of the two fatty acidsis an unsaturated fatty acid and more preferably, at least one of thetwo fatty acids is a precursor to DHA or EPA. ALA is a particularlypreferred unsaturated fatty acid.

In another aspect, the invention comprises a lipid compositioncomprising a plurality of triacylglycerols, wherein a substantial numberof the triacylglycerols comprise an antioxidant moiety, which ispreferably an alpha-keto acid, and more preferably, pyruvic acid. Thefatty acid composition of the lipid composition is preferably primarilyunsaturated, with a ratio of unsaturated to saturated fatty acids ofgreater than about 7 being more preferred. A high proportion ofunsaturated precursors to DHA or EPA, such as ALA, is also preferred.

In one aspect, the invention comprises a method of producing a lipidcomposition comprising the step of interesterifying a source oil ortriacylglycerol mixture with pyruvic acid in molar excess, using alipase catalyst. In a preferred embodiment, the source oil is avegetable oil high in unsaturated content, such as perilla oil, flaxseedoil, hempseed oil, canola oil, or soybean oil, or mixtures thereof.Perilla oil and flaxseed oil, in particular, are known to have a highALA content.

DETAILED DESCRIPTION

The present invention provides for a lipid composition comprisingtriacylglycerols incorporating a non-fatty carboxylic acid as anantioxidant moiety. A lipid composition comprising such triacylglycerolsmay be produced by interesterification of an antioxidant, which may be anon-fatty keto-carboxylic acid, with an oil containing predominantlytriacylglycerols. This invention also comprises a compound blend lipidcomposition, containing predominantly a family of noveltriacylglycerols. It is believed that this composition can provide amultitude of health benefits. When describing the present invention, allterms not defined herein have their common art-recognized meanings.

As used herein, the term “triacylglycerol” is synonymous withtriglyceride and means an ester of glycerol (propane-1,2,3-triol) inwhich all three hydroxyl groups are esterified with a carboxylic acid.As used herein, an “antioxidant moiety” means a moiety which prevents orreduces oxidation of the triacylglycerol.

As used herein, the term “keto acid” refers to a molecule which is botha ketone and an acid. In most keto acids of interest to the presentinvention, the carbonyl group is on the carbon atom adjacent thecarboxyl terminal carbon atom as shown in the formula

wherein the keto acid is alpha-ketoglutaric acid if R is propanoic acidand wherein the keto acid is pyruvic acid if R is methyl as shown in theformulae:

As used herein, the term “fatty acid” refers to saturated or unsaturatedaliphatic monocarboxylic acids which typically have an even number ofcarbon atoms. A long chain fatty acid has over 12 carbon atoms. A mediumchain fatty acid has between 8 to 12 carbon atoms and a short chainfatty acid has between 3 to 6 carbon atoms. A “non-fatty carboxylicacid” refers to any carboxylic acid which is not a fatty acid. Examplesof non-fatty carboxylic acids include dicarboxylic acids, aromatic acidsand keto acids.

In general terms, the invention may comprise a triacylglycerol havingthe formula:

wherein:

at least one of R1 or R3 is an antioxidant alpha-ketocarboxylic acidresidue esterified to the glycerol backbone; and

the remainder of R1, R2 or R3 is a long-chain fatty acid residue.Preferably, the long-chain fatty acid is selected from the groupconsisting of C16 and C18 saturated and unsaturated fatty acids.

In one embodiment, the saturated fatty acid residues are esterified toglycerol in the primary positions as R1 or R3, opposite the antioxidantmoiety. Unsaturated fatty acids may be selected from any of the threeclasses of C18 fatty acids: ω-3, ω-6 and ω-9 fatty acids.

In a preferred embodiment, the ratio of ω-3: ω-6 fatty acids is at least2:1 on a molar basis, more preferably at least about 2.5:1, and mostpreferably greater than about 3:1. The ratio of unsaturated to saturatedfatty acids is preferably greater than about 4 on a molar basis, andmore preferably greater than about 7.

In a preferred embodiment of the lipid composition, a substantialproportion, but not necessarily a majority, of the triacylglycerolsinclude the antioxidant. It is sufficient that enough triacylglycerolmolecules include an antioxidant such that the antioxidant concentrationin the lipid composition is greater than about 10 mol %, preferablygreater than about 20 mol % and more preferably greater than about 30mol %. However, it is not intended here to prescribe a minimumconcentration of the antioxidant as even a small quantity of theantioxidant may have a beneficial effect. It is also preferred, but notessential, that the unsaturated fatty acids are predominantly ALA, witha concentration is the range of greater than about 10 mol %, morepreferably greater than about 20 mol % and most preferably greater thanabout 35 mol %. In one particularly preferred embodiment, theantioxidant concentration is about 35 mol % with an ALA concentration ofabout 50 mol %.

The term “mole percent” or “mol %” refers to the number of moles of onecomponent present in a total of 100 moles of all components. A preferredtriacylglycerol of the invention has an antioxidant alpha-keto acid inthe sn-1 position, ALA and other saturated or unsaturated fatty acids inthe sn-2 and sn-3 positions. If the sn-3 fatty acid is unsaturated, itis preferred that it be a C18, ω-3, ω-6 or ω-9 fatty acid.

The antioxidant acids useful for this invention include but are notrestricted to alpha keto acids such as pyruvic and alpha-ketoglutaricacids. The endowment of stability by these antioxidant acids isreflected in a relatively low peroxide value of 6.5 meq/kg lipid. Theperoxide value is a measure of the level of hydroperoxides present inthe lipid. These hydroperoxides are generally the first products oflipid oxidation; and therefore, the peroxide concentration is a directmeasure of oxidative lability of the lipid.

Another aspect of the invention comprises a method of providing verylong chain ω-3 polyunsaturated fatty acids such as DHA and EPA throughtheir more stable precursors in form of acyl constituents of the lipid.Provision of precursors is also a superior way of providing the DHA andEPA, as their metabolism is less efficient than that of ALA. Theconversion of ALA into DHA and EPA will then take place in a regulatedmanner in the body, as is well-known in the art.

Provision of keto-acid antioxidants and polyunsaturated fatty acids onthe same molecule is believed to enhance the stability of the latter, aswell as mitigate the effect of n−3 fatty acids on the inhibition of someglycolytic pathways. The esterification of both these components ontothe glycerol backbone also enhances the biosorptivity and stability ofthe keto-acids. Keto-acids are relatively small molecules and are easilyabsorbed as compared to the long-chain fatty acids. They typically donot contribute caloric value to a diet. Rather, by virtue of beingintermediates of key energy-yielding reactions in the glycolysis and TCAcycles, they are a source of relatively instant energy enabling furthernutritional benefits.

The lipid composition of the invention may be made by any procedurecommonly used to make structured lipids generally. A survey oftechniques for producing specific-structured triacylglycerols may befound in Xu, “Production of specific-structured triacylglycerols bylipase-catalyzed reactions: a review”, Eur. J. Lipid Sci. Tech. 2000,287-303, the contents of which are incorporated herein. For example,esterification reactions of free fatty acids (in a certain proportion)and pyruvic acid/ester with glycerol, interesterification reactions(transesterification/acidolysis) of fatty acid esters or acylglycerolsof oils with keto-acids or their esters, hydrolysis of lipids followedby reesterification of diacylglycerols with pyruvic acid/ester, etc. aresome useful techniques. Using such methods, the end product is not ahomogenous composition of a single triacylglycerol; the end product willbe a lipid composition comprising many different triacylglycerols.Depending on the reactants used, the catalyst/enzyme that is used (ornot used) and reaction conditions, a host of acylglycerols may beproduced including but not restricted to the mixture of triacylglycerolsand other acylglycerols (di- and mono-acylglycerols) described in theinvention. All of the mixtures and structured lipids formed from themethods herein are within the scope of this invention.

For interesterification, the source oil may comprise mixtures ofsubstantially pure triacylglycerols including ALA, linoleic acid, oleicacid in appropriate proportions. In a preferred embodiment, the sourceoil may be a naturally-occurring oil rich in unsaturated fatty acids,and ALA in particular, such as perilla oil (65%), flaxseed oil (50%),hempseed oil (25%), canola (9%), soybean oil (6-8%), amongst others. Theantioxidant moiety can be introduced in form of its methyl or ethylesters, or as free acid.

Pyruvic acid is stabilized by bonding to the glycerol backbone. Itsbioavailability is ensured by its esterification at the primary hydroxylpositions (sn-1, sn-3), which are the sites of action for pancreatic andgastric lipases. Incorporation of pyruvic acid at the desired positioncan be achieved by judicious selection of lipases with respect to theirselectivity/preference for active group position, substrate size,substrate unsaturation and position of unsaturation in substrate. Theuse of a lipase with a greater preference for a lower degree ofunsaturation also ensures that more of the saturated fatty acids getknocked off the lipid substrate than unsaturated fatty acids. Use oflipases with primary hydroxyl preference would enableesterification-precisely in either of these positions. As a result, in apreferred embodiment, relatively high ratios of unsaturated fatty acidsto saturated fatty acids, and n−3 to n−6, can be maintained. A suitablelipase may include that from Carica papaya latex which is obtained in anaturally-immobilized form. Other suitable lipases may be sourced fromAspergillus niger, Candida lipolytica, Humicola lanuginosa, Mucorjavanicus, Rhizomucor miehei, Penicilium sp., Rhizopus sp., orPsuedomonas sp. Pancreatic lipases or pre-gastric esterases may also besuitable. Reactions are carried out using suitable solvent media. Thechoice of a suitable lipase is well within the skill of one skilled inthe art, given the source oil to be used, the fatty or non-fatty acidsto be incorporated, and the desired characteristics of the resultinglipid composition.

Purification of certain triacylglycerols or types thereof, if desired,can be achieved by standard techniques such as solvent evaporation, thinlayer chromatography (TLC), column chromatography, preparativehigh-performance liquid chromatography (prep-HPLC), supercritical fluidchromatography (SFC), short-path distillation, as is well known in theart. Free fatty acids formed may be removed by distillation or otherappropriate means.

The lipid composition of the present invention may be added as anutraceutical supplement to prepared foods. 5-6 g of designer lipid perday (3 servings) should provide an adequate amount for fulfilling thedaily requirements of the antioxidant and ALA of a typical person. Thiscan be provided in form of dietary supplements or formulated intoyoghurts and other cultured dairy products, nutrient bars, or any otherfoodstuff to which a lipid composition may be added. The resulting lipidhas little color of its own (absorbance=0.052 at 480 nm). This makes thelipid more versatile for various delivery formulations such as yoghurtswhere it has no discernable effect on color or pH (change of 3%) andlittle impact on yoghurt viscosity (change of 10%) for lipidconcentrations of up to 3% (w/w) in yoghurt.

The following examples are intended only to be exemplary of the claimedinvention and not to be restrictive or limiting in any manner.

EXAMPLE I Interesterification

In one embodiment, a lipid composition of the present invention was madeby interesterification of flaxseed oil with pyruvic acid, in a 1:5 molarratio and catalyzed by an immobilized sn-1,3 regioselective lipase fromRhizomucor miehei. The interesterification was carried out at 50° C. forabout 7 hours.

Phase separation of the reaction contents yielded a pyruvic acid richaqueous phase which also contains the lipase and a top organic phasecontaining the reacted and unreacted lipids. The oil phase was purifiedby preparative thin-layer chromatography (TLC) on silica gel G platesusing a solvent mixture of hexane, diethyl ether and acetic acid(70:30:1) as the mobile phase. As a result, the lipids were fractionatedinto triacylglycerols (78%), diacylglycerols (9%), monoacylglycerols(4%) and free fatty acids (9%).

The triacylglycerol fraction was scraped from the plate and eluted withwater saturated diethyl ether, which was then evaporated and theremaining lipid collected. Unreacted pyruvic acid and the immobilizedlipase may be recycled for reuse.

Pyruvate content and fatty acid composition of the acylglycerols wereanalyzed by spectrophotometry and gas chromatography, respectively.

Spectrophotometry

The sample was dissolved in ethanol (for oil samples) or deionized water(for aqueous samples) to give a total volume of 2 ml. 18 ml of deionizedwater were further added and the contents of the vial vortexed. To 1 mlof the above, was added 1 ml of 0.125 g/L dinitrophenylhydrazine (DNPH)reagent and 1 ml deionized water. The vial contents were incubated at37° C. for 10 min, after which 5 ml of 0.6N sodium hydroxide were added,again followed by vortexing. Absorbance was read at a wavelength of 420nm on a Beckmann Diode Array spectrophotometer.

Pyruvate concentration in the sample was then calculated by comparingthe absorbance with that of a standard sample of pyruvic acid of knownconcentration.

Gas Chromatrogrphy (GC)

Fatty acid analysis was carried out by converting all fractions intotheir methyl esters. The TLC fractions are redissolved in 1 ml diethylether and methylated by addition of 50 μL 0.2N methanolic(m-trifluoro-methylphenyl) trimethylammonium hydroxide (TMTH) per 50 mginitial lipid and allowed to stand under ambient conditions for 20-30minutes and injected onto the gas chromatograph after adding methylmyristate as an internal standard. The gas chromatograph used was aHewlett Packard HP5890 equipped with a flame ionization detector (FID).Fatty methyl esters were separated on a 0.25 μm BPX70 column (25 m×0.32mm i.d.) using helium as the carrier gas and temperature programming asfollows: 2 min at 35° C., followed by heating at 7° C./min to 250° C.,and finally for 1.5 min at 250° C. Injections were splitless and theinjector and detector temperatures were 250° C. and 300° C.,respectively. Peak areas and percentages were calculated using anintegration pack using response factors.

Fatty acid concentrations were calculated by comparing the peak areawith that of the internal standard.

Results

95% of pyruvate incorporated was found to occur into thetriacylglycerols (TAG) and the remainder into diacylglycerols (DAG).

TABLE 1 Pyruvate distribution in the reaction system after 7 h Pyruvicacid (%) Initial pyruvate 100 TAG pyruvate 18.43 DAG pyruvate 5.32Unused pyruvate 76.08 Reusable pyruvate 71.57

TABLE 2 Triacylglycerol and diacylglycerol long-chain fatty acylcomposition after 7 h mol % Palmitic Stearic Oleic Linoleic ALA TAG 8.00 4.28 21.57 15.17 50.99 DAG 10.79 4.78 32.44 19.73 32.27

EXAMPLE II Formulation of Lipid Product into Yoghurt

In this example, a lipid composition was prepared so as to yield apyruvic acid concentration of 7.6% w/w lipid (30 mol %). The ALA contentwas 40% w/w lipid (48 mol %). Three different yoghurts were used forformulation purposes: plain, soy and bumbleberry yoghurt. Lipid wasstirred into the yoghurt to yield different concentrations ranging from0 to 3%) and a study on the physical characteristics of the fortifiedand unfortified yoghurts was conducted.

Peroxide value: 65% less than that of the parent oil.

Color & Texture: Unaffected at all concentrations.

Taste and odor: Masking of these properties entirely in the bumbleberryyoghurts. In the regular and soy yoghurts, an alteration in both tasteand odor of yoghurts was noticed beyond 1% lipid.

Temp Viscosity Viscosity Yogurt Type Lipid (%) pH (° C.) (mPa · s/g)Change (%) Regular: No Fat 0.00 3.98 5 23.06 100.00 25 3.00 100.00 372.28 100.00 Regular: No Fat 0.74 4.01 5 23.61 102.39 25 3.43 114.29 372.63 115.13 Regular: No Fat 0.99 3.97 5 25 2.77 92.39 37 2.18 95.51Regular: No Fat 2.00 3.93 5 25 3.10 103.18 37 2.44 107.03 Regular: NoFat 3.01 3.92 5 24.49 106.21 25 3.21 106.99 37 2.51 110.08 Soy: Regular0.00 3.96 5 26.12 100.00 25 4.21 100.00 37 3.73 100.00 Soy: Regular 0.773.95 5 25 5.12 121.70 37 4.32 115.78 Soy: Regular 1.02 3.94 5 25 4.85115.34 37 4.22 113.07 Soy: Regular 1.98 3.92 5 25 4.92 116.87 37 4.20112.66 Soy: Regular 2.80 3.86 5 26.25 100.51 25 4.61 109.57 37 3.73100.00 Fruit: No Fat 0.00 3.83 5 34.91 100.00 (Bumbleberry) 25 4.78100.00 37 3.67 100.00 Fruit: No Fat 0.78 3.82 5 (Bumbleberry) 25 6.62138.51 37 3.32 90.51 Fruit: No Fat 0.96 3.81 5 (Bumbleberry) 25 5.36112.11 37 4.52 123.00 Fruit: No Fat 2.00 3.81 5 (Bumbleberry) 25 5.35111.85 37 4.38 119.27 Fruit: No Fat 3.01 3.78 5 33.14 94.93(Bumbleberry) 25 5.26 109.96 37 4.30 117.20 Parent oil 100.00 5 10.89100.00 25 4.74 100.00 37 3.59 100.00 Pure lipid 100.00 5 10.62 97.55 254.68 98.68 37 3.42 95.25References:

The following references are referred to herein by number and areincorporated herein as if reproduced in their entirety.

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As will be apparent to those skilled in the art, various modifications,adaptations and variations of the foregoing specific disclosure can bemade without departing from the scope of the invention claimed herein.The various features and elements of the described invention may becombined in a manner different from the combinations described orclaimed herein, without departing from the scope of the invention.

1. A triacylglycerol comprising at least one unsaturated long chainfatty acid ester residue having greater than 12 carbon atoms and anantioxidant ester comprising a non-fatty carboxylic acid residue.
 2. Thetriacylglycerol of claim 1 wherein the non-fatty carboxylic acid is anα-keto acid.
 3. The triacylglycerol of claim 2 wherein the α-keto acidis pyruvic acid or α-ketoglutaric acid.
 4. The triacylglycerol of claim1 wherein the at least one long-chain fatty acid comprises an omega-3fatty acid.
 5. The triacylglycerol of claim 4 further comprising along-chain fatty acid which is either saturated or unsaturated.
 6. Thetriacylglycerol of claim 4 wherein the omega-3 fatty acid is a precursorof docosahexanoic acid or eicosapentaenoic acid.
 7. The triacylglycerolof claim 6 wherein the omega-3 fatty acid is α-linolenic acid.
 8. Atriacylglycerol comprising a α-keto acid in the sn-1 or sn-3 position,α-linolenic acid in the sn-2 position and saturated or unsaturated C18fatty acid in the sn-1 or sn-3 position not occupied by the α-keto acid.9. The triacylglycerol of claim 8 wherein the α-keto acid comprisepyruvic acid or α-ketoglutaric acid.
 10. A triacylglycerol of theformula:

wherein R1 is a keto acid residue and R2 and R3 are long chain fattyacids resudues.
 11. The triacylglycerol of claim 10 wherein R3 comprisesan unsaturated fatty acid residue.
 12. The triacylglycerol of claim 11wherein the unsaturated fatty acid residue comprises an omega-3 fattyacid residue which is a precursor of docosahexanoic acid oreicosapentaenoic acid.
 13. The triacylglycerol of claim 12 wherein theomega-3 fatty acid residue is an α-linolenic acid residue.
 14. Thetriacylglycerol of claim 10 wherein R1 is an α-keto acid residue, R2 isa saturated or unsaturated long chain fatty acid residue and R3 is anα-linolenic acid residue.